The proposed research seeks to define completely the growth requirements of several types of cultured human diploid cells. Clonal growth assays will be used to evaluate both the nutritional adequacy and the freedom from toxicity of experimental media. The requirement for serum protein will be reduced as far as possible by improvements in the culture technique and environment, and by qualitative and quantitative optimization of the basal medium. Macromolecular factors that continue to be required will then be isolated and characterized. The goals of the proposed research are to understand fully the environmental requirements for sustained multiplication in culture of various types of human diploid cells, and to develop practical media for such growth that contain only precisely defined components. Substantial progress has already been made toward these goals for fibroblast-like cells derived from human fetal lungs and newborn foreskin. Studies are also in progress with human epidermal keratinocytes, which can now be grown without a feeder layer. Initial emphasis will be on gaining a complete understanding of the requirements of the cells now under study and on development of conditions that will allow detailed study of cells of epithelial morphology that are very difficult to grow with currently available media and techniques. Later emphasis will be on gaining a detailed understanding of the growth requirements of human epithelial cells and also of cells that exhibit differentiated properties in culture. The media that will be developed during this study will greatly facilitate research in many different areas of human cell biology and biomedical science.